Protease inhibitors have emerged as a powerful drug class1. They include the inhibitors of angiotensin converting enzyme, inhibitors of HIV proteases and proteasomal inhibitors such as Bortezomib (Velcade) used to treat multiple myeloma2.
The proteases of the endo-lysosomal pathway have frequently been proposed as therapeutic targets as they play important roles in the regulation of a wide variety of biological systems3. For example, lysosomal cysteine and aspartyl proteases are validated drug targets in several trypanosome species4 and the upregulation of certain endosomal proteases is associated with increased malignancy5. Asparagine endopeptidase (AEP or legumain) has also been implicated in the progression of malignant melanoma6, in the destruction of the therapeutic drug 1-asparaginase and in neuroexitotoxity7. Down-regulation of cystatins, which are natural cysteine protease inhibitors, can lead to increased malignancy8 and faulty immune responses9. High expression of Cathepsin D (Cat D) in non-Hodgkin's lymphoma has also been associated with increased malignancy10 and is also associated with poor prognosis in breast cancer11. A further potential therapeutic application of endosomal protease inhibitors would be immune modulation since several recent studies demonstrate that the proteolytic activity in endosomes of antigen presenting cells may be too high leading to antigen destruction and inefficient presentation to T-cells. Consequently, protease resistant antigens often elicit more robust immune responses12,13.
Taken together, it seems that effective down-modulators of endo/lysosomal protease activity could be a valuable addition to the therapeutic armoury. However, to date modulation of endo/lysosomal protease function has remained challenging, as there are multiple families of endosomal proteases with an extensive functional redundancy14. As an additional problem, there is evidence in the literature that the knock-down/inhibition of specific proteases leads to the upregulation of others3, 15.
Most endosomal proteases belong to 3 distinct families: There are several papain-like cysteine proteases (PLCPs), including cathepsin L,S,B,C and several others16. Alongside these there are the aspartyl proteases related to pepsin; cathepsins D and E. Finally, there is an additional cysteine protease termed asparaginyl endopeptidase (AEP) or legumain which is more closely related to the caspases17. Each of these 3 classes can be inhibited by distinct and non-overlapping small molecule inhibitors18, but in vivo inhibition, or knock-out, of these proteases frequently shows limited or no phenotype most likely due to functional redundancy. We thus postulated that inhibiting all three families of endosomal proteases in an endosome-specific manner would provide a powerful tool for modulating endo/lysosomal function.
PLCPs and AEP are potently inhibited by a naturally occurring 14 kDa protein, cystatin C. The cystatins are a family of small proteins, that inhibit PLCPs with sub-nanomolar affinity19. They are present in the bloodstream and are believed to play a role in the mopping up of proteases released during physiological and pathological responses. Importantly cystatin C, as well as several family members, inhibit AEP via a distinct binding site with a Ki of 0.20 nM20 (FIG. 1). Cystatin C thus represents an excellent scaffold for the synthesis of a pan-endosomal protease inhibitor.
Cathepsin D and E, the endosomal aspartyl proteases are inhibited with a Ki of 0.1 nM by pepstatin A21, an isopeptide first isolated from Actinomyces. Its major drawback is its virtual insolubility in aqueous media21. Nonetheless it is still widely used even in cell based assays because more soluble alternatives are not readily available. Several attempts have been made to address this problem, such as conjugating pepstatin A to asialoglycoprotein(ASGP)22, or to poly(ethylene glycol)23, or more recently to directly mannosylate it, or conjugate it to mannosylated bovine serum albumin24. PEG-ylation of Pepstatin reduces its inhibitory potential 400-fold, and conjugating to mannosylated BSA-reduces the Ki 10-fold, whereas conjugation to ASGP renders pepstatin inactive until the protein backbone is digested. Conjugation of pepstatin to peptides or fluorescent moieties did not significantly alter its inhibitory potential25.